Smooth muscle membrane vesicle orientation: a study on intactness and sidedness of rat myometrium plasma membrane vesicles

Plasma membrane vesicles of rat myometrium were prepared in media containing 240 mM sucrose. The vesicles were exposed to isotonic, hypertonic, and hypotonic sucrose concentrations, fixed, sectioned, and studied using the electron microscope. The vesicles fixed in isotonic media were circular in appearance. Vesicles fixed in hypertonic media were distorted and showed a reduced volume to surface ratio consistent with the hypothesis that >80% of the vesicles were osmotically active to sucrose. Cationized ferritin binding studies and Ca binding and release studies were also consistent with this finding. Exposure to hypotonic media also yielded membranes with distorted profiles indicating that they had been ruptured. [3H]Sucrose trapping experiments revealed that the vesicles had an internal volume of 1.20–1.44 mL/g protein. Hypotonic shock treatment reduced this intravesicular volume to 0.20–0.28 mL/g protein. The hypotonic shock treatment also led to enhanced galactose oxidase catalyzed Na3B3H4 labelling of the membranes and to increased K+-activated ouabain-sensitive p-nitrophenyl phosphatase activity. The enhancement was the same (55 ± 10%) in the various membrane preparations for both the parameters. The data are interpreted to conclude that the rat myometrium plasma membrane vesicles consisted of 20% broken vesicles and equal proportions of intact vesicles of inside-out and rightside-out orientations.

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The iodide channel of the thyroid: a plasma membrane vesicle study

AJP Cell Physiology ◽

10.1152/ajpcell.1992.263.3.c590 ◽

1992 ◽

Vol 263 (3) ◽

pp. C590-C597 ◽

Cited By ~ 45

Author(s):

P. Golstein ◽

M. Abramow ◽

J. E. Dumont ◽

R. Beauwens

Keyword(s):

Plasma Membrane ◽

Chloride Transport ◽

Membrane Vesicle ◽

Membrane Vesicles ◽

Anion Channel ◽

Apical Plasma Membrane ◽

Plasma Membrane Vesicle ◽

Plasma Membrane Vesicles ◽

Bovine Thyroid ◽

Set Up

The uptake of radioactive iodide or chloride by plasma membrane vesicles of bovine thyroid was studied by a rapid filtration technique. A Na(+)-I- cotransport was demonstrated. When this Na(+)-I- cotransport is inactive (i.e., at 4 degrees C and in the absence of Na+), an uptake of iodide above chemical equilibrium could be induced, driven by the membrane potential. The latter was set up by allowing potassium to diffuse into the membrane vesicles in the presence of valinomycin and of an inward K+ gradient. This potential difference (positive inside) induced the uptake of iodide (or other anion present). The data support the existence of two anionic channels. The first one, observed at low near-physiological iodide concentration (micromolar range), which exhibits a high permeability and specificity for iodide (hence called the iodide channel), has a Km of 70 microM. The other one appears similar to the epithelial anion channel as described by Landry et al. (J. Gen. Physiol. 90: 779-798, 1987); it is still about fourfold more permeable to iodide than to chloride and presents a Km of 33 mM. Under physiological conditions the latter channel would mediate chloride transport, and the iodide channel, which is proposed to be restricted to the apical plasma membrane domain of the thyrocyte, transports iodide from the cytosol to the colloid space.

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Fetal lung epithelial cells contain two populations of amiloride-sensitive Na+ channels

AJP Lung Cellular and Molecular Physiology ◽

10.1152/ajplung.1993.264.4.l357 ◽

1993 ◽

Vol 264 (4) ◽

pp. L357-L364 ◽

Cited By ~ 4

Author(s):

S. Matalon ◽

M. L. Bauer ◽

D. J. Benos ◽

T. R. Kleyman ◽

C. Lin ◽

...

Keyword(s):

Plasma Membrane ◽

Membrane Vesicles ◽

Fetal Lung ◽

Lung Epithelial Cells ◽

Na Channel ◽

Na Transport ◽

Plasma Membrane Vesicles ◽

Binding Studies ◽

Channel Protein ◽

Lung Epithelial

Active Na+ transport by the alveolar epithelium plays a major role in reabsorption of the fetal lung fluid after birth. We characterized the biochemical and physiological characteristics of Na+ conductive pathways in distal fetal lung epithelial (FLE) cells isolated from 20-day-old rat fetuses. We demonstrated that a polyclonal antibody to Na+ channel protein (NaAb) binds to the plasma membranes of FLE cells. In Western blot studies, this NaAb and an anti-idiotypic monoclonal antibody to the amiloride-binding subunit of the Na+ channel protein recognized 150- and 90-kDa polypeptides in plasma membrane vesicles of FLE. 22Na+ flux measurements across plasma membrane vesicles of FLE revealed the existence of electrogenic Na+ transport, which was twice as high as the corresponding adult value. One hundred micromolars of amiloride, benzamil, and 5-(N-ethyl-N-isopropyl)-2'-4'-amiloride inhibited 30, 40, and 70% of the electrogenic Na+ transport across plasma membrane vesicles of FLE cells, respectively. The half-maximum inhibition of electrogenic Na+ transport by these substances occurred between 0.3 and 1 microM. [3H]benzamil equilibrium binding studies in membrane vesicles of FLE cells revealed the existence of two binding sites that had dissociation constant values of 19 and 1,525 nM, respectively. These data indicate the presence of both high- and low-amiloride affinity Na+ conductive pathways (channels) in FLE cells.

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In vitro release of physically separable factors from monocytes that exert opposing effects on erythropoiesis

Blood ◽

10.1182/blood.v67.5.1454.1454 ◽

1986 ◽

Vol 67 (5) ◽

pp. 1454-1459 ◽

Cited By ~ 6

Author(s):

L Feldman ◽

CM Cohen ◽

N Dainiak

Keyword(s):

Plasma Membrane ◽

Membrane Vesicle ◽

In Vitro Release ◽

Membrane Vesicles ◽

Plasma Membrane Vesicles ◽

Wide Range ◽

Dose Dependent Fashion ◽

Membrane Bound ◽

Opposing Effects

Abstract In order to investigate the capacity of monocytes to release erythroid burst-promoting activity (BPA), we added media conditioned by homologous monocytes to both serum-free human and serum-restricted murine marrow culture. We found that soluble, membrane vesicle-free culture medium is a potent source of the growth factor. On the other hand, monocyte membranes or exfoliated plasma membrane vesicles elaborate a factor that inhibits erythroid burst formation by up to 100%. Inhibitory activity is expressed in a dose-dependent fashion over a wide range of concentrations (0.001 to 10 micrograms/mL) tested. Experiments with antilymphocyte plasma membrane IgG, which has been shown to neutralize both soluble and membrane-bound lymphocyte-derived BPA in human marrow culture, indicate that the expression of soluble BPA by monocytes is unaffected by these antibodies. Furthermore, while antimembrane IgG is capable of absorbing BPA from LCM supernatants, these antibodies are ineffective in removing BPA from MCM supernatants, suggesting that these two soluble growth factors may be antigenically distinct. Our findings indicate that while monocytes release soluble BPA, they are also a source of membrane-associated factors that exert inhibitory effects on erythropoiesis in vitro.

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Neutrophil plasma membranes. I. High-yield purification of human neutrophil plasma membrane vesicles by nitrogen cavitation and differential centrifugation.

The Journal of Cell Biology ◽

10.1083/jcb.86.1.21 ◽

1980 ◽

Vol 86 (1) ◽

pp. 21-28 ◽

Cited By ~ 82

Author(s):

M S Klempner ◽

R B Mikkelsen ◽

D H Corfman ◽

J André-Schwartz

Keyword(s):

Plasma Membrane ◽

Human Neutrophil ◽

Plasma Membranes ◽

Hydrolytic Enzymes ◽

Membrane Vesicles ◽

Galactose Oxidase ◽

High Yield ◽

Differential Centrifugation ◽

Plasma Membrane Vesicles ◽

Equilibrium Ultracentrifugation

Neutrophil chemotaxis, phagocytosis, and oxygen-dependent microbicidal activity are initiated by interactions of stimuli with the plasma membrane. However, difficulties in neutrophil plasma membrane isolation have precluded studies on the precise structure or function of this cellular component. In this paper, a method is described for the isolation of representative human neutrophil plasma membrane vesicles, using nitrogen cavitation for cell disruption and a combination of differential centrifugation and equilibrium ultracentrifugation in Dextran gradients for membrane fractionation. Multiple biochemical markers and galactose oxidase-tritiated sodium borohydride surface labeling were employed to follow the yield, purity, and distribution of plasma membranes, nuclei, lysosomes, endoplasmic reticulum, mitochondria, and cytosol. According to these markers, neutrophil plasma membranes were exposed to minimal lysosomal hydrolytic enzymes and could be isolated free of other subcellular organelles. In contrast, disruption of neutrophils by mechanical homogenization resulted in > 20% lysosomal rupture and significant plasma membrane proteolysis. Electron microscopy demonstrated that plasma membranes isolated after nitrogen cavitation appeared to be sealed vesicles with striking homogeneity.

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In vitro release of physically separable factors from monocytes that exert opposing effects on erythropoiesis

Blood ◽

10.1182/blood.v67.5.1454.bloodjournal6751454 ◽

1986 ◽

Vol 67 (5) ◽

pp. 1454-1459

Author(s):

L Feldman ◽

CM Cohen ◽

N Dainiak

Keyword(s):

Plasma Membrane ◽

Membrane Vesicle ◽

In Vitro Release ◽

Membrane Vesicles ◽

Plasma Membrane Vesicles ◽

Wide Range ◽

Dose Dependent Fashion ◽

Membrane Bound ◽

Opposing Effects

In order to investigate the capacity of monocytes to release erythroid burst-promoting activity (BPA), we added media conditioned by homologous monocytes to both serum-free human and serum-restricted murine marrow culture. We found that soluble, membrane vesicle-free culture medium is a potent source of the growth factor. On the other hand, monocyte membranes or exfoliated plasma membrane vesicles elaborate a factor that inhibits erythroid burst formation by up to 100%. Inhibitory activity is expressed in a dose-dependent fashion over a wide range of concentrations (0.001 to 10 micrograms/mL) tested. Experiments with antilymphocyte plasma membrane IgG, which has been shown to neutralize both soluble and membrane-bound lymphocyte-derived BPA in human marrow culture, indicate that the expression of soluble BPA by monocytes is unaffected by these antibodies. Furthermore, while antimembrane IgG is capable of absorbing BPA from LCM supernatants, these antibodies are ineffective in removing BPA from MCM supernatants, suggesting that these two soluble growth factors may be antigenically distinct. Our findings indicate that while monocytes release soluble BPA, they are also a source of membrane-associated factors that exert inhibitory effects on erythropoiesis in vitro.

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Sodium-gradient-stimulated transport of l-alanine by plasma-membrane vesicles isolated from liver parenchymal cells of fed and starved rats. Crucial role of the adrenal glucocorticoids

Biochemical Journal ◽

10.1042/bj2080685 ◽

1982 ◽

Vol 208 (3) ◽

pp. 685-693 ◽

Cited By ~ 12

Author(s):

Dennis C. Quinlan ◽

C. Gordon Todderud ◽

Darshan S. Kelley ◽

Rolf F. Kletzien

Keyword(s):

Plasma Membrane ◽

Active Transport ◽

Membrane Vesicle ◽

Membrane Vesicles ◽

Plasma Membrane Vesicle ◽

Plasma Membrane Vesicles ◽

Free System ◽

Liver Parenchymal ◽

Alanine Transport ◽

Parenchymal Cells

The ability of liver efficiently to take up amino acids, particularly l-alanine, during starvation was studied in a cell-free system by isolating plasma-membrane vesicles in a transport-competent state from rat liver parenchymal cells. These membrane vesicles have the capacity to accumulate l-alanine against an apparent concentration gradient when exposed to an artificial and transient transmembrane Na+ gradient (extravesicular Na+ concentration greater than inside). The rate of accumulation of l-alanine is dependent on the plasma-membrane vesicle concentration, and the steady-state concentration attained is inversely related to the osmolarity of the medium. The Na+-mediated stimulation is not exhibited if the membrane vesicles are pre-equilibrated with NaCl, if K+ or Li+ are substituted for Na+, or if SO42− replaces Cl− as the counterion. The apparent active transport of l-alanine into the membrane vesicles appears to occur by an electrogenic mechanism: (1) the use of NaSCN significantly heightens the early concentrative phase of transport when compared with the effect of NaCl; (2) an enhanced active transport is also observed when a valinomycin-induced K+ efflux occurs concomitant with Na+ and l-alanine influx. Plasma-membrane vesicles isolated from liver parenchymal cells of a 24 h-starved rat exhibit an initial l-alanine transport rate that is 3–4 times that for membrane vesicles derived from a fed animal. The increased rate of l-alanine transport by plasma-membrane vesicles from starved animals can be obliterated by adrenalectomy and restored by administration of glucocorticoid. These results establish that stimulation of the gluconeogenic pathway by starvation involves a plasma-membrane-localized change affecting l-alanine transport which is regulated in part by the glucocorticoid hormones.

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